The ability to measure the mechanical properties of tissue and materials in vivo with high spatial resolution would have a broad range of biomedical applications. Conventional mechanical testing has been widely used, but is invasive and difficult to apply to biological samples in vivo, whereas acoustic techniques such as ultrasound and elastography are not well suited for quantitative measurement requiring high sensitivity and accuracy. The objective of this program is to develop a new method based on Brillouin light scattering to probe the biomechanical properties, non-invasively and quantitatively with microscopic resolution. Brillouin scattering is an inelastic process mediated by acoustic phonons, similar to Raman scattering by vibrational phonons. The first specific aim of this proposed study is to develop a Brillouin microscope by combining a novel high-extinction fully-parallel spectrometer with high-resolution confocal imaging. The feasibility of this instrument in tissue engineering and ophthalmology will be tested. The second specific aim is to measure the viscoelastic moduli of cross-linked collagen scaffolds for cartilage repair. Biomechanical microenvironments within collagen-cell constructs will be imaged over time with cellular resolution. The third specific aim is to measure the viscoelastic properties of the crystalline lens, cornea, and vitreous in mice and monitor their age-dependent changes in vivo longitudinally. A successful demonstration may open up a new possibility for understanding and evaluating various age-related ocular problems in vivo. Measuring tissue mechanical properties in vivo and with high spatial resolution remains a significant challenge. The ability to measure and image the biomechanical properties of tissue and biomaterials in vivo by Brillouin microscopy would have broad applications in tissue engineering, cataract surgery, drug delivery, intraoperative tissue characterization, as well as basic investigations to understand the role of tissue biomechanics in various diseases and age-related problems.